Budding yeast Saccharomyces cerevisiae (“S. cerevisiae”) is one of the most important organisms in biotechnology. An enormous number of studies have been performed, and currently there are two types of regulated promoters in the yeast. The first type is an innate yeast promoter, such as the set of GAL promoters, expression of which is repressed by glucose and activated by galactose. The GAL1 promoter is used in many cases because its induction ratio is very high, but the high concentration of galactose required in the system can be problematic. Apart from the GAL promoters, researchers can use repressive promoters from the MET3 gene (negatively regulated by methionine) and PHO5 (negatively regulated by inorganic phosphate). However, the use of these promoters has multiple potentially undesirable effects on metabolism and host gene transcription and, importantly, can lead to a slow growth rate or a high cost incompatible with biotechnological applications. In other words, they are suboptimal, especially for biotechnology applications.
The other type of promoters consists of synthetic functional units derived from other organisms, such bacteria and viruses. One of the most studied switches, called the Tet system, has been applied to regulate expression in yeast. In that system, a transcription factor, the TetR protein from Escherichia coli (“E. coli”), can bind to its operator sequence depending on the presence or absence of tetracycline or derivative compounds such as anhydrotetracycline or doxycycline. However, use of the antibiotics hinders large-volume fermentation in industry because of their expense and moreover the use of the antibiotics is undesirable from a regulatory standpoint. Thus, a low-cost alternate system is desirable to facilitate regulated protein and pathway expression for yeast fermentation in a large-scale of millions of liters at one time. In addition, an increase in the number of available ligand-activated switches provides more options for fundamental and applied studies. The vast number of known and unknown promoters, however, makes it very difficult to identify possible new candidates that would overcome the problems of the existing systems.
Previous articles have described an autoregulated camphor oxidation operon in the 240-kb plasmid PpG1 from Pseudomonas putida (“P. putida”). Expression of the enzymes in this operon is induced by the presence of camphor, because a TetR-homolog transcription factor, camR, when bound to camphor dissociates from the bound operator. Notably, camphor is very inexpensive and widely used, even in human daily life. None of the literature, however, has shown that the autoregulated camphor operon of P. putida would be active in S. cerevisiae or other systems.